A critical step in the HIV life cycle is transcription of the HIV genome directed by the transactivator protein Tat. Transactivation by Tat requires the cellular cofactor, TAWP-TEFb, which is composed of the catalytic subunit Cdk9 and regulatory subunit cyclin T1. TAK/P-TEFb is recruited by Tat to the HIV promoter and increases processivity of transcriptional elongation by modifying RNA polymerase II. This application seeks to further elucidate molecular mechanisms of Tat and TAK/P-TEFb function and to increase understanding of how Tat and TAK/P -TEFb activity are regulated. The application builds on recent findings that Cdk9 and cyclin T1 are present in the nucleoplasmic and the splicing factor-enriched speckle compartments of the nucleus, and the functional implications of this result. The proposed approach is based on the emerging awareness of the importance of the link between gene expression and nuclear organization, and relies heavily on recent advances in high resolution microscopy techniques, as well as biochemical and genetic techniques. State-of-the-art cell biology techniques have no1 previously been exploited for proteins involved in Tat transactivation, and will be used to address aspects of Tat and TAWP-TEFb function within the host cell that can not readily be answered using other approaches. The hypothesis to be tested in this proposal is that TAK/P -TEFb functions to link transcription elongation and mRNA processing/transport, and that the subnuclear localization of Cdk9 and cyclin T I is regulated, dynamic and important for TAK/P -TEFb function. Furthermore, Cdk9 and cyclin T1 localization may be altered by Tat and/or other viral gene products during HIV infection to allow more efficient expression of the HIV genome. To test these hypotheses, the specific aims of this application are: (1) to investigate the function and regulation of cyclin T1 and Cdk9 in association with the nuclear speckle compartment; (2) to examine the spatial relationships between TAWP-TEFb, HIV gene products, and subnuclear compartments in primary and cultured cells; and (3) to analyze promoter recruitment of Cdk9, cyclin T1, and other factors by Tat and additional activators at the single cell level. It is anticipated that results of these experiments will provide new mechanistic insights into the function of Tat and TAK/P-TEFb, as well as provide information regarding HIV pathogenesis at the subcellular level. This information may form the basis for the rational design of novel antiviral strategies.